1. Field of the Invention
This invention relates to methods of extracting and purifying bioactive substances from various plants and herbs. More specifically the invention relates to methods of extracting and separating bioactive substances from various plants and herbs using supercritical fluid extraction and/or fluorocarbon solvent extract. The present invention further relates to separation of bioactive substances contained in extracts using packed column supercritical fluid chromatography. The present invention also relates to formulations, pharmaceutical preparations and dietary supplements which may be prepared with the extracted bioactive substances and use of such pharmaceutical preparations and dietary supplements to treat various human ailments.
2. Description of the Background
Throughout history humans have ingested and otherwise consumed a wide variety of plants and herbs, and extracts of such plants and herbs to help alleviate aches and pains, improve immunity to infection, treat various illnesses, or even to induce relaxation or stress reduction.
One plant that has been commonly ingested by the people of the South Pacific to induce relaxation is called Kava Root. K. Schubel, J. Soc. Chem. Ind., 43, 766 (1924); A. G. Van Veen, Rec. Trav. Chim., 58, 52 (1939). Kava root consists of the dried rootstock and/or shoots of Piper methysticum Forst (Family: Piperaceae). The Kava root is most typically ingested by drinking an aqueous macerate (pulverized Kava root mixed with water) known as the beverage Kava.
First attempts to identify the active compounds within Kava root were made over a hundred years ago. Those efforts resulted in the identification of kavalactones, also known as kavapyrones. More than ten kavalactones as well as four other substances have been identified in the Kava root to date, including kavain, dihydrokavain (a.k.a. marindinin), methysticin, dihydromethysticin, yangonin, and desmethoxyyangonin. V. Lebot, M. Merling, and L. Lindstrom, xe2x80x9cKava the Pacific Drugxe2x80x9d, Yale University Press, New Haven, Conn. (1992). These compounds are neutral, nitrogen-poor compounds that may be specifically referred to as substituted d-lactones and substituted a-pyrones. The lactone ring is substituted by a methoxy group in the C3 position, and the differences in the compounds lie in the degree of unsaturation (e.g. yangonin, desmethyoxyyangonin, kavain and methysticin) or by bezene substitution (e.g. dihydrokavain and dihydromethysticin), as shown in FIG. 24.
The particular kavalactones in a Kava root extract vary depending upon its origin. Different species of kavalactones have been found to have varying physiological effects in vivo depending on their molecular structure. All naturally occurring kavalactones contain an enolic double bond between C3 and C4. The dienolides of the yangonin type appear to be pharmacologically inert. In the enolides, the effective optimum varies as a function of the hydrogenation of the double-bonded C7. For example, kavain has the strongest effect as a local anesthetic, dihydromethysticin as a spasmolytic, and dihydrokavain as an intensifier of narcosis. R. Hansel, Characterization and Physiological Activity of Some Kava Constituents Pacific Science, July 1968, Vol. XXII: pp293-313.
Further, the particular kavalactones present depend upon whether, in addition to rhizome parts, roots and stems of the plant are included in the extract. High quality extracts of the Kava root are sold based upon the total kavalactone content, rather than upon analysis of the individual lactones contained therein. The concentration ranges of total kavalactone levels in the Kava root extracts employed, e.g. in Germany are generally within the range of 30 to 55 weight percent.
Although many types of kavalactones have been identified, no simple and efficient method is available for both extraction of the root and separation of each individually extracted lactone. The traditional extraction method (e.g. steam distillation) usually involved mixing 100 grams of root with a suitable quantity of distilled water producing a slurry having a volume of approximately 200 mL. A. R. Furgiuele, W. J. Kinnard, M. D. Aceto, and J. P. Buckley, J. Pharmaceutical Sci., 54, 248 (1965). The slurry was steam distilled and the first 100 mL of distillate was collected, filtered and lyophilized. The yield for each extraction was about 50 mg. Alternately, a liquid-solid extraction at room temperature has been reported wherein the above slurry was intimately mixed in a Waring blender for 15 minutes. The mixture was then filtered and lyophilized. In certain cases, rather than lyophilization, the filtrate was subjected to successive extractions with chloroform. This purification operation basically removed impurities from the aqueous layer. The extraction yield for these methods varied depending on the solvent and methodology used.
Modern Kava root extracts are commonly manufactured using ethanol as a solvent because kavalactones arc readily soluble in ethanol. The extractable materials are in the form of a yellowish brown paste or powder, which is then tested to assure proper concentrations of kavalactones.
A plant that has been commonly ingested by the people of Mexico and other Latin American countries is Byrsonima crassifolia (Nanche). The medicinal importance of this tropical tree, which is indigenous to Mexico, has been documented historically since the sixteenth century. Traditional healers use the plant to treat gastrointestinal disorders, especially diarrhea and dysentery.
To date, about 21 chemical substances have been extracted from the dried leaves and bark of the tree, including xcex2-sitosterol and betulin (triterpenes), pipecolic acid and proline (amino acids), and catechin and quercetin (flavonoids). Bxc3xa9jar, E., et al., Constituents of Byrsonima crassifolia and their spasmogenic activity, Int. J. Pharmacog. 1995, 33:1, 25-32. The discovery of pipecolic acid is significant in that it is a rare compound in nature and is an important intermediate in a number of pharmacological preparations which demonstrate therapeutic effect for stroke, Parkinson""s disease, Alzheimer""s disease, and other. neurological and vascular disorders. Prior to the discovery of pipecolic acid in Byrsonima crassifolia, preparations containing pipecolic acid were derived from various cultured micro-organisms.
Traditional healers prepared aqueous solutions of Byrsonima as teas. It was recently discovered that aqueous extracts of Byrsonima contain only catechin. However, when methanol is used to extract bioactive substances from Byrsonima, a wide variety of triterpenes, amino acids and flavonoids can be isolated.
Plants in the genera Aesculus and Crataegus are known to contain bioactive substances which affect the heart and circulatory system. Galenical preparations of, for example, Crataegus oxyacantha, C. azarolus, C. monogyna, C. pentagyna, C. laevigata and C. nigra have been used in European herbalism for centuries for these purposes. Crataegus pinnatifida has been used for similar purposes in Traditional Chinese Medicine for even longer. Likewise the use of Aesculus hippocastanum in Europe for the treatment of circulatory disorders is well documented. The effect has been attributed to aescin, a mixture of triterpene glycosides which have an anti-exudative and vascular tightening effect. While these European and Asian species have been the subject of a great deal of research, co-generic species endemic to the New World have been largely ignored. Aesculus californica, commonly known as xe2x80x98California buckeyexe2x80x99 in English and xe2x80x98berrucoxe2x80x99 in Spanish, had been used by the native tribes and early colonists of California for a variety of purposes. The dried bark of the tree was used for toothaches, the fresh seeds were eaten after leaching out the bitter principles, and the unprocessed fruits were used to treat hemorrhoids, as a fish poison, and as an abortifacient.
Analyses of the seeds of Aesculus californica by several groups have revealed the presence of a number of known bioactive compounds: the proteids xcex2-methyl alanine, phenylalanine, isohomoleucine, isohomo-6-hydroxyleucine, mino4-methyl-hex-tans-4-enoic acid and gamma-glutamyl-2-A-hex-4-enoic acid; the benzoids arbutin and hydroquinone; the flavonoid epicatechin; and the coumarin eleutheroside B-1, as well as the carbohydrate quebrachitol. This chemical profile differs from the European A. hippocastanum. 
Extracts of Crataegus and Aesculus species are commonly prepared using various solvents, such as methanol, ethanol or acetone. The extracts are taken from the leaves and flowers of Crataegus species and from the seeds, leaves and bark of the Aesculus species.
The plant Simmondsia chinensis, also known as Jojoba, is native to the desert areas of the Southwestern United States and Mexico. Jojoba has a unique wax ester oil which is 50 to 60% of its seed weight. This oil is currently used in cosmetics and lubricants. The remainder of the seed is not used as much as the oil although it contains about 25% crude protein after the oil is removed. The defatted meal contains sugars and 11 to 15% of a unique group of natural products.
Simmondsin, one of the natural products contained in Jojoba meal, has been shown to be an effective hunger satiation agent by reducing food intake in mice, rats, and chickens. Cokeleare et al. (1995, Ind. Crops Prod., 4:91-96). Simmondsin has also been shown to be a useful weight reduction agent for Dogs. See U.S. Pat. No. 5,962,043. However, Jojoba meal also contains other antinutritional factors such as trypsin inhibitor, polyphenols, bitter taste, nonnutritive protein, and indigestible Jojoba oil.
Methods of removing so-called xe2x80x9ctoxicxe2x80x9d principles from Jojoba seed meal in order to render it palatable to animals as feed have been described. See U.S. Pat. No. 5,672,371 to d""Oosterlynck, U.S. Pat. No. 4,209,534 to Banigan et al., and U.S. Pat. No. 4,148,928 to Sodini. Also, solvents have been used to extract simmondsins from Jojoba meal. U.S. Pat. No. 6,007,823.
Pfaffia paniculata, commonly called Brazilian ginseng, is a plant in the family Amaranthaceae which grows in parts of Brasil, Paraguay, Uruguay and Argentina. All parts of the plant are used in folk medicine, but it is the roots that are considered most valuable medicinally. Traditionally, the plant has been used to treat diabetes, rheumatism, ulcers, leukemia and other cancers, and as a tranquilizer, general tonic, and aphrodisiac.
Recent studies have demonstrated that the plant has biological activity as an anti-allergenic, analgesic, anti-inflammatory, antitumor agent, has a weak CNS-depressant effect and decreases vascular permeability. The plant has further been shown to be non toxic to humans.
Extracts of the plant have been shown to contain allantoin, daucosterol, b-ecdysone, pfaffic acid, pfaffosides A, B, C, D, E, and F, polypodine B, xcex2-sitosterol, stigmasterol, and stigmasterol-3-O-xcex2-D-glucoside.
Tumera diffusa and other Turnera species, commonly called damiana, hierba del venado, and other names, are small, herbaceous perennials ranging from California to South America. The plant has been used since pre-Columbian times as an aphrodisiac and sexual tonic, expectorant, diuretic, antidiabetic, to increase fertility, treat spermatorrhea, orchitis, nephritis, chronic coughing, and as a stimulant, digestive aid, and laxative. Laboratory tests of various Turnera preparations have shown cytotoxic and antihyperglycemic effects. The plant extract has been found to be non-mutagenic.
Turnera species are known to contain arbutin, caffeine, gonzalitosin, xcex2-sitosterol, an acetovanillin-like benzenoid compound, hexacosan-1-ol, tetraphyllin B, N-triacontane, tricosan-2-one, an essential oil which contains 1-8-cineol, paracymene, a-pinene, b-pinene, and three sesquiterpenes.
The roots of plants of the genus Perezia produce perezone (2-(1,5-dimethyl-4-hexenyl)-3-hydroxymethyl-p-benzoquinone). Perezone is a sesquiterpenic benzoquinone which exhibits oxido-reduction characteristics. Certain species of the perezia genus have been used as laxatives in Mexican folk medicine.
In studies of the effect of perezone on electron transport in biological membranes, it was found that perezone inhibits mitochondrial electron transport in rat liver mitochondria differently than rotenone, amytal, and Antimycin A. Carabez A. et al., The Action of the Sesquiterpenic Benzoquinone, Perezone, on Electron Transport in Biological Membranes. Arch Biochem Biophys. 1988 January; 260(1):293-300. The low respiration of rat liver mitochondria depleted of coenzyme Q10 (CoQ) was shown to be increased by perezone.
Heimia salicifolia was used as a traditional medicine in the Americas to treat inflammation. In recent studies, two alkaloids from Heimia salicifolia, cryogenine and nesodine, were discovered to be more than twice as potent as aspirin as inhibitors of prostoglandin synthetase prepared from bovine seminal vesicles.
In-vitro-grown shoots of Heimia salicifolia have been found to be active in alkaloid biosynthesis, yielding the biphenylquinolizidine lactones vertine, lytrine, and lyfoline, the ester alkaloids demethoxyabresoline and epidemethoxylabresoline, the phenylquinolizidinols demethyllasubine-I and demethyllasubine-II. Rother, A., The phenyl- and biphenyl-quinolizidines of in-vitro-grown Heimia salicifolia. J. Nat. Prod. 1985 January-February; 48(1):3341. Five to ten day old seedlings of Heimia salicifolia have also been used to extract bioactive species. Two isomeric 2-hydroxy-4-(3-hydroxy4-methoxyphenyl) quinolizidines, differing in the configuration of the bridgehead carbon, have been isolated by Rother, A. et al. Radioactive dilution has been used to isolate 2-keto-4-(3-hydroxy4-methoxyphenyl)quinolizidine from the seedlings.
Although these and many other plant species are known for various therapeutic and healing effects, these plants have further benefits, and synergistic effects when multiple plants are combined, that have not yet been described. The bioactive substances which make these plants medicinally effective are commonly extracted with solvents and/or water. This technology has several disadvantages among which are the cost of the solvents, costs associated with their safe disposal, and removal of the solvents from the extract.
Furthermore, medicinal plant chemistry is complex and the vast majority of medicinal plants owe their pharmacological action to many different molecular entities which often belong to more than one class of compounds. Many solvents have only a limited effectiveness for eluting certain classes of compounds, resulting in inefficient extractions. These types of extractions generally result in low concentrations of bioactive substances and a need for multiple extractions with different solvents to isolate differing substances.
An extraction method which removes high concentrations of multiple bioactive substances is desirable. A separation method which permits efficient""separation of the substances to obtain purified, therapeutically effective quantities of bioactive substances is also desired. Such methods would provide new extracts from known plant species, the ability to isolate useful quantities of specific bioactive substances, new uses of extracts from known plant species, and more efficient extraction.
Supercritical fluid extraction and supercritical fluid chromatography have been used in the chemical arts for many years. Gases such as carbon dioxide or propane have proven to have excellent solvating properties when pressurized, particularly above their critical point This so-called supercritical region occurs when a gas is pressurized to a point where it would normally liquify, but is simultaneously heated above its now greatly reduced boiling point to prevent liquification. This xe2x80x9csupercritical fluidxe2x80x9d is neither a liquid nor a gas, but exhibits properties of both. In particular, supercritical fluids possess excellent solvating properties with high selectivity for particular analytes. This selectivity can be further adjusted by variations of pressure, temperature and use of mixed gases.
Lopez and Benedicto used supercritical CO2 to extract kavalactones from Kava herb. V. Lopez-Avila and J. Benedicto, J. High Resolut. Chromatogr., 20, 555 (1997). In each extraction a 10 mL cartridge was filled with 2.5 grams of Kava herb which was extracted with both pure and 15% ethanol-modified CO2 for a dynamic extraction time of 60 minutes at 250 atm and 60xc2x0 C. Extracted analytes were collected in a vial filled initially with 4 mL of ethanol maintained at 22xc2x0 C. Recovery was less than 25% when pure CO2 was used as the extraction fluid, but was greater than 90% (relative to a solid-liquid extraction) when using 15% ethanol-modified CO2. Identification of each of the extracted kavalactones was determined via GC/MS. Not only was the supercritical fluid extraction highly efficient, but there were very few co-extractives.
Although CO2 proved generally effective for extraction of kavalactones, CO2 only works as an extraction medium at extreme pressures, generally on the order of several thousands of pounds per square inch. This factor contributes to the high cost of equipment and to inherent dangers associated with extreme pressure vessels. Various types of chromatography have been used to separate and determine the major constituents of Kava extracts. Nakayama et al. used thin layer chromatography to separate and quantify six kavalactones (R. L. Young, J. W. Hylin, D. L. Plucknett, Y. Kawano, and R. T. Nakayama. Phytochemistry, 5, 795 (1966)). Later, Gracza et al. used normal phase high pressure liquid chromatography (HPLC) to separate a mixture of kavalactones (L. Gracza and P. Ruff, J. Chromatogr., 486, 193 (1980)). Haberlein et al. have also used normal phase HPLC to separate and quantify a series of kavalactones (H. Haberlein, G. Boonen, and M. A. Beck; Planta Med. 63, 63 (1997); G. Boonen, M. A. Beck, and H. Haberlein, J. Chromatogr. B, 702, 240 (1997)). Reverse phase HPLC was used to separate kavalactones, however, most of the separations were poor. R. M. Smith, H. Thakrar, T. A. Arowolo, and A. A. Shafi J. Chromatogr., 283, 303 (1984). Recently, Shao et al. used reverse phase HPLC with atmospheric pressure chemical ionization mass spectrometry in the positive ion mode to separate and identify specific kavalactones. Baseline separation of six lactones was achieved in less than 36 minutes. Y. Shao, K. He, B. Zheng, and Q. Zheng. J. Chromatogr. A, 825, 1 (1998).
Although some of these methods have proven fairly efficient for identifying, obtaining, separating, and isolating various kavalactones, improvements to the field are necessary. Additionally, a method for simply and accurately obtaining, separating and isolating different species of bioactive substances from other plant species are still lacking. Furthermore, in today""s health conscious society, novel applications of natural source substances, and methods for obtaining such therapeutically useful substances, are necessary.
The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides novel methods for extracting, separating, and isolating bioactive substances from natural sources. The present invention further relates to novel therapeutic uses of such extracts.
Accordingly, one embodiment of the invention is directed to methods for the preparative and/or commercial scale extraction of bioactive substances comprising the step of using supercritical fluid extraction (SFE) or near-critical extraction (NCE) for said preparative and/or commercial-scale extraction. The SFE or NCE may be accomplished with CO2 or CO2 modified with various other volatile substances. The SFE or NCE may further be accomplished as a batch-wise extraction, continuous-cascading extraction, or countercurrent-solvent extraction.
Another embodiment is directed to methods for the preparative and/or commercial scale processing of bioactive substances comprising coupling SFE or NCE and supercritical fluid chromatography (SFC), with or without modifiers, for said preparative and/or commercial scale processing. In this embodiment, isopropyl amine may be used as a modifier in SFC.
Another embodiment of the invention is directed to methods for the preparative and/or commercial scale extraction of bioactive substances comprising the step of using dense gases in the supercritical, near critical, or subcritical state with or without modifiers, for said preparative and/or commercial scale extraction. The dense gas may be any non-chlorinated fluorocarbon solvent and the modifiers may be any other volatile substance. The extraction may be performed under a pressure of 0-10 bar, or under supercritical or near critical fluid conditions. Dense gas extraction may further be accomplished as a batch-wise extraction, continuous-cascading extraction or countercurrent-solvent extraction
Another embodiment of the invention is directed to methods for the separation of bioactive substances comprising the step of SFC. The step of using SFC preferably comprises the use of HH2 and/or C4 columns, singly or in combination, in the SFC separation.
Another embodiment of the invention is directed to compositions comprising medicinal formulations of extracts of Byrsonima species recovered with supercritical fluid extraction and/or dense gases or with various organic solvents and/or water, and to methods of administering therapeutically effective amounts of these formulations to patents in need of treatment. Byrsonima species extracts are used alone or are combined with advantageous effect with various Psidium and Enterolobium species extracts, which are similarly prepared. Compositions may comprise extracts or isolated products of Aesculus californica and Crataegus mexicana, either on their own, in combination with one another, or in combination with extracts from various Bursera species.
Another embodiment of the invention is directed to extraction of simmondsin compounds from Jojoba (Simmondsia chinensis) and use of these compounds as a human weight loss agent.
Another embodiment of the invention is directed to formula and compositions comprising a combination of extracted phytochemicals from Turnera species and Pfaffia species, with or without muira puama (a crude drug derived from various species including Ptychopetalum olacoides, Liriosma ovata, and Chaunochiton kappleri) for use as a health tonic and to support sexual function.
Another embodiment of the invention is directed to formula and compositions comprising a combination of extracted phytochemicals from, for example, Heimia salicifolia, for use as a Non-steroidal Anti-inflammatory Drug (NSAID).
Other embodiments and advantages of the invention are set forth in part in the description which follows, and in part, will be obvious from this description, or may be learned from the practice of the invention.